As known, lactic acid bacteria (LAB) are intensively used in the dairy industry for making different animal milk fermented products such as e.g. yogurt, cheese etc.
Vitamin K is important for a number of human/animal health issues such as bone health.
The lactic acid bacteria Lactococcus lactis and Leuconostoc lactis are natural producers of vitamin K2 [menaquinones] (Morishita T et al. 1999. Production of Menaquinones by Lactic Acid Bacteria. J. Dairy. Sci. 82:1897-1903).
The vitamin K2 is constituents of the bacterial plasma membrane—i.e. vitamin K2 is present in the membrane of the LAB.
Generally speaking, the amount of vitamin K2 produced by wild-type lactic acid bacteria is not sufficiently high to make a commercially relevant product comprising vitamin K2—e.g. a dairy product with a sufficiently high amount of vitamin K2.
Accordingly, work has been done to identify mutants of Lactococcus lactis capable of producing an increased amount of vitamin K2—see e.g. WO2008/040793A1 (Danone).
Further, in WO2008/040784A1 (Danone) it is described that an increased amount of produced vitamin K2 can be obtained by fermenting milk with LAB under conditions, where the LAB are not in the growth phase but in what is termed “resting cells” phase in WO2008/040784A1 (see e.g. claim 1).
Essentially, this is obtained by adding a relatively large amount of LAB to the milk.
As known in the art, when a relatively high amount of LAB is added to the milk there will virtually be no growth (cell division) of the LAB, due to the fact that the initially added large amount of LAB will rapidly produce so much lactic acid that the pH of the milk will drop to a pH e.g. below pH=4 and at this low pH there is no significant growth of the LAB.
Said in other words, when a large amount of LAB is added to milk the pH will drop so rapidly that the LAB do not have “time” for making growth (cell division).
As known to the skilled person the thyA gene is encoding thymidylate synthase. As shown in FIG. 1, when this thyA gene is inactivated in e.g. a LAB one gets a so-called thymidine auxotrophic mutant—i.e. a mutant strain that is not capable of cell division in a thymidine “limited” media due to it cannot synthesize dTTP without addition of thymidine and thereby cannot replicate their genome. As known, milk does not comprise a significant amount of thymidine and such thyA(−) mutant strain will therefore not have significant growth in milk.
WO00/01799A2 (Chr. Hansen A/S) describes the thyA(−) Lactococcus lactis mutant strain MBP71 deposited under the accession number DSM12891 and it is described that the thyA(−) strain is bacteriophage resistant.
It is not described or suggested that such a thyA(−) mutant strain could be used for making an increased amount of vitamin K2.
Further, in none of above and below cited prior art documents is it described or suggested that by inactivating the thyA gene in a LAB cell one could get increased vitamin K2 production.
For instance, the above discussed WO2008/040793A1 (Danone) mentions a number of genes that could be relevant for obtaining increased vitamin K2 production in LAB (see e.g. claim 5)—however none of these mentioned genes are the thyA gene or genes that in the present context may be said to be similar/related to the thyA gene.
JP 2001 136959 A (published May 2001) relates to a Bacillus subtilis microbial cell culture useful for preventing and treating osteoporosis, whereby said microbial cells are recovered before releasing vitamin K. The osteoporosis treating effect can be obtained by ingesting the microbial cell containing vitamin K, whereby said microbial cells can be incorporated in food or pharmaceutical product (cf. abstract).
JP 2000 080043 A (published March 2000) concerns oral pharmaceuticals/foodstuffs comprising a water soluble vitamin K fraction of Bacillus natto for the prevention of osteoporosis (cf. abstract).
The article (Tsukamoto et al; “Construction of a Bacillus subtilis (natto) with High Productivity of Vitamin K2 (Menaquinone-7) by Analog Resistance”; Bioscience Biotechnology Biochemistry; vol. 65(9), pages 2007-2015, 2001) reports on the construction of a Bacillus subtilis strain with high productivity of vitamin K2 (menaquinone-7/MK7). Hereby a strain OUV23481 with two-fold higher productivity of MK7 than that of a commercial strain is constructed as a mutant with analog resistance to 1-hydroxy-2-naphtoic acid (HNA), p-fluoro-D,L-phenylalanine (pFP), mfluoro-D,L-phenylalanine (mFP) and beta-2-thienylalanine (betaTA). Said mutant is classified as Basillus subtilis natto (cf. abstract, 2nd paragraph of the right-hand column on page 2007 and 2nd paragraph of left-hand column on page 2014).